Biosynthesis of C-phycocyanin trimers

Phycobiliproteins, particularly C-phycocyanin (CPC), serve as major light-harvesting complexes in cyanobacteria, exhibiting high efficiency in light-harvesting and energy transfer. Currently, the easy and large-scale acquisition of functional CPC remains a major challenge, primarily due to the requirement for precise, sequential covalent attachment of multiple phycocyanobilin (PCB) chromophores. In this study, by using a dual-promoter (T7 and araBAD) system to control the sequential binding of two PCB chromophores to β82 and β153, we successfully achieved the biosynthesis of β-CPC (λ_{max, absorption} = 605 nm, λ_{max, emission} = 644 nm) in E. coli, which can transfer energy from the β153-PCB to the β82-PCB. The assembly of α-CPC with PCB-β82, PCB-β153, and PCB₂-β under identical conditions indicates that only PCB₂-β, which covalently binds the two PCB chromophores, can assemble with α-CPC to form a complete CPC trimer (λ_{max, absorption} = 617 nm, λ_{max, emission} = 646 nm). The structure of the assembled trimer reveals that it adopts a typical phycobiliprotein fold, with multiple chromophores precisely arranged, exhibiting features highly similar to those of native CPC. Furthermore, we established a biosynthetic pathway for CPC trimers in E. coli. This system provides a powerful tool for engineering phycobiliproteins with various light-harvesting and energy transfer properties, facilitating future studies on artificial photosynthesis, light-harvesting antenna design, and the fundamental mechanisms of excitation energy transfer.